Lipid-based nanoparticles such as, e.g., liposomes, nano- and microemulsions, SLN (solid lipid nanoparticles), nanocapsules, nanospheres and lipoplexes are important auxiliaries for a multitude of technical processes and medical applications.
Liposomes are spherical entities consisting of lipides. In aqueous solutions liposomes are formed by a self-aggregation of said lipides with the formation of a lipid double layer, said lipid double layer enclosing an aqueous interior space.
Depending on physical parameters such as mechanical effects, pressure, temperature and the ion concentration and the present lipides and auxiliaries, unilamellar, oligolamellar or multilamellar liposomes are formed. Depending on the components thereof, said liposomes may carry a positive or negative excess charge.
Liposomes may also be loaded with substances which, depending on the lipophilia or hydrophilicity thereof, are predominantly enclosed in the lipid layer or predominantly enclosed in the aqueous interior of the liposomes. Such liposomes are used in diagnostic detection processes, as therapeutic agents for transporting active substances within the organism or as an active substance depot. In addition, said liposomes may also be used in the biological and biomedical research and in plant protection (e.g., for the transport of substances into cells). Also a use in cosmetics is possible.
The properties of liposomes such as, e.g., the stability or storability thereof, essentially depend on the substances existing in the lipid layer.
For the manufacturing of liposomes, membrane-forming lipids such as, e.g., phosphatidylcholine, phsophatidylglycerol, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, sphingomyelin, cationic lipides such as, e.g., DOTAP, DC-Chol, DOTMA, Inter alia, membrane-forming amphiphiles such as, e.g., block polymers, alkyl esters, alkyl ethers, alkyl amides of sugars, diols and polyols, amines, amino acids, peptides and sugars and cholesterol and other substances are used.
For the manufacturing of empty or substance-loaded liposomes being as uniform as possible various processes are known. These processes have been reviewed by Lasch et al. in a concise manner (Lasch, J. et al, Preparation of Liposomes; in “Liposomes—a practical approach”, Torchilin, V. P. and Weissig, V., Ed., 2nd edition (2003)).
Many manufacturing processes start from so-called “hand-shaken vesicles” which may be formed by a simple rehydration of a lipid film and a subsequent shaking (In most cases in a flask). In most cases, said liposomes are very non-uniform with respect to the size and the lamellarity thereof. The liposome size may be standardized (usually reduced in size) and the lamellarity of said liposomes may be reduced using processes such as extrusion, the freeze-thaw method or ultrasonication:
Extrusion processes use an extruder and usually enable the manufacturing of only small amounts (manual extrusion) (Macdonald, R. E. et al., BBA 1061:297-303 (1991)). The manufacturing of large amounts involves high expenditures in equipment (pump, membranes etc.) and a cumbersome processing. Moreover, only low-viscosity media can be extruded resulting in a low inclusion efficiency. Due to the open manipulation of media, the preparation of sterile samples is cumbersome, and with radioactive substances there is a danger of a contamination of the environment. Moreover, prior to extrusion and hydration lipid mixtures have to be produced by a common dissolution and a subsequent evaporation.
Although injection processes and detergent processes are possible in industrial scale, the removal of solvents and detergents is problematic. High lipid concentrations cannot be obtained in a simple way, and the excess of aqueous media allows only low inclusion efficiencies to be achieved with hydrophilic substances.
Also freeze-thaw steps may standardize the size of non-uniform, often multilamellar liposomes and lower the lamellarity thereof. In most cases, an increased inclusion efficiency for water-soluble substances arises.
With the above-mentioned processes in particular the low enclosing efficiency for hydrophilic substances is a problem inherent in the system. The reason for this is that said processes can be carried out with only low lipid amounts and consequently the formed liposomes can enclose only a low portion of the aqueous total volume. Consequently, only low amounts of hydrophilic substances dissolved in the aqueous phase will be enclosed.
The enclosing efficiency may be increased, e.g., by a liposome manufacturing using high-pressure homogenization since considerably larger lipid amounts may be used in this case. As a result, a so-called liposome gel having a very high lipid content is obtained, wherein the aqueous outer volume approximately corresponds with the aqueous inner volume with respect to the order of magnitude. Then, the content of the enclosed hydrophilic active substance is correspondingly high. Another advantage is the possibility to manufacture high formulation amounts which is easily achieved using high-pressure homogenization. Moreover, the high pressure homogenization is advantageous in that it produces especially small vesicles which are especially interesting in the medial field, e.g., for a tumour targeting using the so-called EPR effect (enhanced permeability and retention). This effect is based on the vascular permeability of blood vessels in tumours which is strongly increased in most cases. Due to the leakiness of the vessels, small particles such as, e.g., liposomes (in particular if they are very small) may leave the vascular bed and enrich in the tumour (Yuan, F. et al., Cancer Res. 54:3352-3356 (1994)).
The economically especially advantageous properties of high-pressure homogenization (small vesicles, high enclosure efficiency, high sample amounts) is connected with a number of drawbacks:
The manufacturing of sterile materials absolutely essential for the use in humans and animals is problematic. Although a sterile manufacturing is possible, it is cumbersome since the necessary high-pressure homogenization has to be performed under clean-room conditions or the material has to be autoclaved subsequently. Moreover, autoclaving vesicular phospholipid gels (VPG) containing active substances often poses problems with the stability of active substances and/or lipides (Moog, R. et al., Int. J. Pharm. 206:43-53 (2000)).
With the use of high-pressure homogenization it is especially difficult to produce small sample amounts required, e.g., in medical or molecular-biological laboratories (if only a small sample amount is available, if radioactive substances are used etc.) or in the screening of a very large number of different lipid formulations (e.g., in the field of preformulation).
Moreover, the homogenization step heavily strains the sample which is critical with expensive and sensitive substances such as, e.g. biological materials (DNA, siRNA, antibodies, peptides, proteins) or with sensitive low-molecular substances such as, e.g., antioxidants, lipids containing specific highly unsaturated fatty acids, cytostatics (alkylating agents etc.).
The equipment required for high-pressure homogenization is expensive, bulky and unacceptable for many (in particular medical/molecular-biological) laboratories. Since each composition change requires the machine to be cleaned, the sample throughput is low; a screening of different mixtures is practically impossible and can be automated only with difficulties (e.g., in the field of preformulations).
Moreover, an outstanding technological know-how is required, not least to limit the danger for the environment resulting from the use of hazardous substances (e.g., radioactive substances or cytostatics).
The problems encountered in the manufacturing of liposomes have already resulted in the use of liposome gels for cosmetic purposes, said gels being formed only by a rehydration of a lipid mixture with a standardization of the vesicles being totally dispensed with (DE 10255285). Such formulations are not suitable for use in pharmacy, biomedicine and medicine and at least critical for use in cosmetics due to a low reproducibility of the vesicle composition.
SLN are nanoparticles having a size of from about 50 to 1000 nm. A review of SLN is given in Pharmazeutische Technologie. Moderne Arzneiformen. R. H. Müller und G. E. Hildebrand, Wissenschaftliche Verlagsgesellschaft Stuttgart, 1998. Contrary to emulsions, the matrix material consists of solid lipides. Here, physiological lipides (e.g., corresponding triglycerides) or lipides from physiological components (e.g., glycerides from endogenic fatty acids) are predominantly used. It is supposed that this achieves a good in vivo compatibility (Weyhers, H. et al., Proc. 1st world meeting APV/APGI, Budapest, 489-490 (1995)). However, the matrix material is not restricted to physiological lipids, also waxes and non-physiological triglycerides being conceivable.
To date, SLN have been manufactured by high-pressure homogenization of water-dispersed lipides in molten or solid states (hot or cold homogenization, see Müller, R. H., Pharm. Ind. 49; 423-427 (1997); EP 0 605 497; Schwarz, C. et al., J. Controlled Rel. 30:83-96 (1994); Müller R. H. et al., Eur. J. Pharm. Biopharm. 41:62-69 (1995)). This manufacturing technique by high-pressure homogenization is characterized in that the SLN size is very homogenous and, moreover, the amount of microparticles is very low. However, as in the manufacturing of liposomes, the expenditure for said high-pressure homogenization is very high.
Another type of lipid-based nanoparticles are droplets in emulsions, with submicron emulsions (SME) being meant here, that is, O/W emulsions having droplet/particle sizes below 1 μm (Benita, S. et al., J. Pharm. Sci. 82: 1069-1079 (1993)). So-called nanoemulsions having droplet sizes of from 50 to 1000 nm cannot be limited therefrom.
Said emulsions have been used in the parenteral feeding for a long time (Lucks, J. S. et al., Krankenhauspharmazie 15:51-57 (1994)), however, they may also be used as excipient. A review covering SME is found in Pharmazeutische Technologie: Moderne Arzneiformen. R. H. Müller und G. E. Hildebrand, Wissenschaftliche Verlagsgesellschaft Stuttgart, 1998.
Among the SME manufacturing processes used today, the high-pressure homogenization (using piston gap homogenizers or a microfluidizer technique) is the leading technique. In the laboratory or pilot plant scale, also ultrasonic homogenization is used mainly because high-pressure homogenization is too tedious.
WO 02/09677 describes the manufacturing of a dispersion comprising a O/W or W/O emulsion and an active substance sparingly soluble in oil and water (amphotericine B). Said dispersion may contain an amount of the active substance exceeding the amount obtained by adding the maximum solubility in water or oil. However, homogenization is high-energetically, that is, by high-pressure homogenization accompanied by the above-mentioned drawbacks. Said process has also been described as the so-called SolEmuls technology (Müller, R. H. et al., Int. 3. Pharm. 269:293-302 (2004)). Here, by using high-energy high pressure homogenization sparingly soluble active substances such as carbamazepin, itraconazoles or amphotericine B are incorporated into emulsions by co-homogenization which results in a strong increase in particular of the dissolution speed but also has the above-mentioned drawbacks of a high pressure homogenization.
Hence, the problem addressed by the invention was to provide a process for the manufacturing of lipid-based nanoparticles kept as simple as possible, said process being milder and safer than high pressure homogenization, suited for screening and enabling the manufacturing of nanoparticles also and in particular in the laboratory scale.